TRICYCLIC ANTIDEPRESSANTS in SERUM and/or in PLASMA by UV CODE Z56010 ( Nordoxepin, Desipramine, Imipramine (TAD1), Doxepin, Amitriptyline and Nortriptyline (TAD2) )
INTRODUCTION
Tricyclic Antidepressants (abbreviation TCA) are a class of antidepressant drugs first used in the 1950s. They are named after the drug’s molecular structure, which contains three rings of atoms. They are used in numerous applications; mainly indicated for the treatment of clinical depression, neuropathic pain, nocturnal enuresis and attention-deficit hyperactivity disorder (ADHD), but they have also been used successfully for headache (including migraine headache), anxiety, insomnia, smoking cessation, bulimia nervosa, irritable bowel syndrome, narcolepsy, pathological crying or laughing, persistent hiccups, interstitial cystitis, ciguatera poisoning, and as an adjunct in schizophrenia.
For many years they were the first choice for pharmacological treatment of depression. Although still considered effective, they have been increasingly replaced by SSRIs and other newer drugs. Newer antidepressants are thought to have fewer side effects and are also thought to be less effective if used in a suicide attempt, as the treatment and lethal doses are farther apart than with the tricyclic antidepressants. They are sometimes still used to treat refractory depression that has failed to respond to standard SSRI therapy.
The exact mechanism of action is not well understood, however it is generally thought that tricyclic antidepressants work by inhibiting the re-uptake of the neurotransmitters norepinephrine and serotonin by neurons. Tricyclic antidepressants may also possess any affinity for muscarinic and histamine H1 receptors to varying degrees, also increase the effects of H1 histamine, and thus most have sedative effects.
Tricyclic antidepressants overdose is a significant cause of fatal drug poisoning. The severe morbidity and mortality associated with these drugs is well documented and due to their cardiovascular and neurological toxicity. Initial or mild symptoms include drowsiness, a dry mouth, nausea and vomiting. More severe complications, include hypotension, cardiac rhythm disturbances, hallucinations and seizures.
EUREKA srl – LAB DIVISION Head Quarter: VAT N° 01547310423 Via Enrico Fermi 25 E-mail:[email protected] 60033 Chiaravalle (AN) ITALY www.eurekaone.com Tel. +39 071 7450790 Fax + 39 071 7496579
This product fulfills all the requirements of Directive 98/79/EC on in vitro diagnostic medical devices (IVD). The declaration of conformity is available upon request. Release N° 001 TAD in serum/plasma by UV July 2012
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TECHNICAL FEATURES
Principle of the Method:
The Tricyclic Antidepressants are isolated purifying the serum/plasma with a clean-up column. After the adding of a stabilization solution the eluate is directly injected into HPLC system.
Recovery : 97 %
Sensitivity : 5 µg/l
Dynamic Range of the Method : 5 - 2000 µg/l
Therapeutic Range in serum : Nordoxepin : 110 – 250 µg/l Desipramine : 75 – 160 µg/l Imipramine : 150 – 250 µg/l Doxepin : 110 – 250 µg/l Amitriptylin : 100 – 250 µg/l Nortriptylin : 50 – 150 µg/l CV%: Nordoxepin : 3,9% intra serie; 4,3% inter serie Desipramine : 4,0% intra serie; 6,1% inter serie Imipramine : 4,5% intra serie; 6,2% inter serie Doxepin : 7,8% intra serie; 7,7% inter serie Amitriptylin : 7,2% intra serie; 7,3% inter serie Nortriptylin : 6,4% intra serie; 6,1% inter serie Components of the kit : All the reagents are ready-to-use and stable 3 years at 2–8 °C. The Calibrators are stable 24 months at 2- 8 °C. Reagent A – Diluting Solution, 1 x 40 ml
Reagent B – Conditioning Solution N° 1, 1 x 300 ml
Reagent C – Conditioning Solution N° 2, 1 x 300 ml
Reagent D – Wash Solution N° 1, 1 x 300 ml
Reagent E – Wash Solution N° 2, 1 x 80 ml
Reagent F – Eluting Solution, 1 x 50 ml
Reagent G – Internal Standard Clomipramine Solution, 1 x 2 ml See Warnings
Reagent H – Test TAD1 Solution, 1 x 2 ml See Warnings
Reagent I – Test TAD2 Solution, 1 x 2 ml See Warnings
Reagent P – Plasma Calibrator lyophil. for Doxepin and Nordoxepin, 1 x 5 ml See Warnings
Reagent Q – Plasma Calibrator lyophil. for Amitriptyline and Nortriptyline, 1 x 5 ml See Warnings
Reagent R – Plasma Calibrator lyophil. for Desipramine and Imipramine, 1 x 5 ml See Warnings
Clean-up Columns, 100 Pcs
Reagent M – Mobile Phase, 5 x 500 ml
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Minimum Instrumental equipment required: Isocratic HPLC System with loop of 100 µl Spectrophotometric Detector λ=216 nm Chromatograms Recorder
Optional Equipment: Autosampler Operational Computer
Whole Blood Collection Procedure : Collect 5 ml of blood in a suitable test tube without gel or with anticoagulant EDTA. Centrifuge at 4000 rpm for 5 minutes. Separate the serum and store at – 20 °C. Stable 4 weeks.
3 PREANALYTICAL PROCEDURE
Preparation of Test Solution.
Pipette in a tube :
• 900 µl of H2O HPLC grade • 100 µl of Reagent H – Test Sol. TAD1 or Reagent I – Test Sol. TAD2 • 20 µl of Reagent G – Internal Standard Sol.
INJECTION :
• Inject 100 µl of this solution in the chromatographic system.
Verify that the Test Solution has retention time similar to fig. 1. If the Test is all right you can start with the analytical procedure; if not, check the functionality of the analytical system.
Important : Don’t use this solution to calibrate!
ANALYTICAL PROCEDURE
STEP 1 : Preparation of Samples, Calibrator and Controls
Pipette in a tube :
Blank Sample Calibrator Water HPLC grade 1000 µl Sample 1000 µl Reagent P or Reagent Q or Reagent R – 1000 µl Calibrator Reagent A – Diluting Sol. 400 µl 400 µl 400 µl Reagent G – Internal Standard Sol. 20 µl 20 µl 20 µl
Vortex for 10 sec.
STEP 2 : Clean-up Columns Conditioning
Prepare a number of Clean-up columns like the number of samples to analyse + the Calibration Standard and the possible Controls.
• Condition clean up columns by fluxing 3 ml of Reagent B – Conditioning Sol. N° 1 and don’t let the columns be dry • Condition clean up columns by fluxing 3 ml of Reagent C – Conditioning Sol. N° 2 and don’t let the columns be dry
STEP 3 : Samples Loading
Dispense 1,420 ml of sample, previously prepared, into Clean-up columns and discharge the percolated liquid, if needed a vacuum system could be used for percolation, at speed of 1 ml / min ( drop by drop ).
Release N° 001 TAD 1 in serum/plasma by UV July 2012
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STEP 4 : Washing
Wash with 3 ml of Reagent D – Washing Sol. N° 1 Let dry with a light flux of air and discharge the percolated liquid
Wash with 0,8 ml of Reagent E – Wash Sol. N° 2 Let dry with a light flux of air and discharge the percolated liquid
STEP 5 : Elution
Elute with 0,5 ml of Reagent F – Eluting Sol. Let dry with a light flux of air and discharge the percolated liquid
• Dilute the eluate with 1 ml of H2O HPLC grade
Vortex for 10 sec.
N.B.: at this step, the sample is stable 2 days at 2-8 °C
INJECTION :
• Inject 100 µl of the eluate into the chromatographic system.
Release N° 001 TAD 1 in serum/plasma by UV July 2012
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TRICYCLIC ANTIDEPRESSANTS - Warnings
REAGENT G : INTERNAL STANDARD CLOMIPRAMINE SOLUTION
The consumption of Clomipramine, contemporaneous at TAD, determinates a wrong quantitative determination.
REAGENT H : TEST SOLUTION TAD1
TRICYCLIC ANTIDEPRESSANTS 1 : Nordoxepin 33,33 mg/l Desipramine 33,33 mg/l Imipramine 33,33 mg/l
REAGENT I : TEST SOLUTION TAD2
TRICYCLIC ANTIDEPRESSANTS 2 : Doxepin 33,33 mg/l Amitriptyline 33,33 mg/l Nortriptyline 33,33 mg/l
REAGENT P : PLASMA CALIBRATOR LYOPHIL. - Lot.N°9226
TRICYCLIC ANTIDEPRESSANTS : Nordoxepin 400 µg/l Doxepin 391 µg/l Use and Reconstitution: Plasma Calibrators are used for calibration of the HPLC system. This lyophilised calibrator has to be prepared like a patient sample. Add exactly 5.0 ml of HPLC water to the vial and mix for 15 min. When all material is dissolved, the solution is ready to use. Stability: 24 months if stored at 2-8 °C. After reconstitution the stability of the analytes is at least 25 days when stored at 2–8 °C. Don’t use after expiry date. Packaging : 1 x 5 ml Warning: The calibrator derives from human plasma, so it could be potentially infected. It must be handled with care.
REAGENT Q : PLASMA CALIBRATOR LYOPHIL. - Lot.N°9224
TRICYCLIC ANTIDEPRESSANTS : Amitriptyline 370 µg/l Nortriptyline 404 µg/l Use and Reconstitution: Plasma Calibrators are used for calibration of the HPLC system. This lyophilised calibrator has to be prepared like a patient sample. Add exactly 5.0 ml of HPLC water to the vial and mix for 15 min. When all material is dissolved, the solution is ready to use. Stability: 24 months if stored at 2-8 °C. After reconstitution the stability of the analytes is at least 30 days when stored at 2–8 °C. Don’t use after expiry date. Packaging : 1 x 5 ml Warning: The calibrator derives from human plasma, so it could be potentially infected. It must be handled with care.
6 REAGENT Q : PLASMA CALIBRATOR LYOPHIL. - Lot.N°9224
TRICYCLIC ANTIDEPRESSANTS : Desipramine 400 µg/l Imipramine 400 µg/l Use and Reconstitution: Plasma Calibrators are used for calibration of the HPLC system. This lyophilised calibrator has to be prepared like a patient sample. Add exactly 5.0 ml of HPLC water to the vial and mix for 15 min. When all material is dissolved, the solution is ready to use. Stability: 24 months if stored at 2-8 °C. After reconstitution the stability of the analytes is at least 30 days when stored at 2–8 °C. Don’t use after expiry date. Packaging : 1 x 5 ml Warning: The calibrator derives from human plasma, so it could be potentially infected. It must be handled with care.
SPECTROPHOTOMETRIC DETECTOR PARAMETERS
λ 216 nm GAIN 0,001 AUFS INTEGRATION TIME 10 sec.
HPLC COLUMN PROTECTION
To save the analytical column Synergi MAX RP 4,6 x 150 mm, 4 µ, the use of Metasaver Precolumn Filter 0.5 um (1 x 10 pcs.) cod. ZA6005 is obligatory.
HPLC COLUMN CONDITIONING
Install a new analytical column Hypersyl Gold CN (4,6 x 150 mm, 5 µ). Disconnect the detector and filter H2O: Acetonitrile ( 20 : 80 v/v ) solution set flow at 1,2 ml / min. Don’t recycle the washing solutions. Filter the mobile phase with a vacuum system and a suitable filter of 0,22 µ. Condition the column with the mobile phase at a flow of 1,2 ml / min. for 20 minutes. Finally inject the Chemical Standard and verify the quality of the HPLC run. It is NOT possible to make analysis at recycling phase. If room temperature is > 20 °C store the Mobile Phase at 2-8 °C between an analytical session and another.
COLUMN CLEANING
Disconnect the detector. Flux a solution made of H2O : Acetonitrile (20 : 80 v/v) for 30 min at a flow of 1,2 ml / min and discharge. Store the column in a solution of H2O : Acetonitrile (20 : 80 v/v). Wash the needle of injection with a solution of H2O : Acetonitrile or Ethanol (80 : 20 v/v).
HPLC PARAMETERS
LOOP 100 µl FLOW 1,2 ml/min. PRESSURE About 90 bar
INTEGRATOR HP – 3394 / 3395 / 3396 PARAMETERS
ATTENUATION 8
OPERATIONAL COMPUTER PARAMETERS
IN CONFORMITY WITH THE SPECIFICATION OF OPERATIONAL COMPUTER SOFTWARE
7 ACCESSORIES AND CONSUMABLES
CODE DESCRIPTION PACKAGING Calibrator for Tricyclic Antidepressant (Imipramine, Z71922 5 x 5 ml Desipramine) Calibrator for Tricyclic Antidepressant (Amitiptyline, Z71924 5 x 5 ml Nortriptyline) Z88116 Calibrator for Tricyclic Antidepressant (Doxepin, Nordoxepin) 5 x 5 ml Control for Tricyclic Antidepressant (Imipramine, Desipramine) Z71923 5 x 5 ml - Level 1 Control for Tricyclic Antidepressant (Amitiptyline, Nortriptyline) Z71925 - Level 1 Z88117 Control for Tricyclic Antidepressant (Doxepin, Nordoxepin) 5 x 5 ml ZTF25805154630 Hypersil Gold CN (150 x 4,6mm -5 um) Analytical Column 1 PK S29057U Standard glass vials of 2 ml with screw cap 1 x 100 PK
8 TRICYCLIC ANTIDEPRESSANTS IN SERUM and/or IN PLASMA ( Reference Chromatograms )
600 100 mV T1+IS.DATA mV PL1.DATA 550 90 500 80 450 400 70
350 60 300 50 250 DOXEPINA 40 200 NORTRIPTILINA 30 150 AMITRIPTILINA
100 IS 20
50 DOXEPINA 10 IS 0 NORTRIPTILINA AMITRIPTILINA -50 0 RT [min] RT [min] 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12
Fig. 1 Test Solution TAD 1 Fig. 2 Plasma Calibrator lyophil. R.T. 6.2 Doxepin R.T. 5.6 Doxepin 150 ug/l R.T. 7.1 Nortriptyline R.T. 6.4 Nortriptyline 150 ug/l R.T. 7.7 Amitriptyline R.T. 7.5 Amitriptyline 150 ug/l R.T. 10.5 Internal Standard R.T. 10.0 Internal Standard
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TRICYCLIC ANTIDEPRESSANTS IN SERUM and/or IN PLASMA ( Reference Chromatograms )
300 300 mV 16 251.DATA mV 20 271.DATA 280 280
260 260
240 240
220 220
200 200
180 180 is
160 160 is
140 140
120 120
100 100
80 80
60 60
40 40 nordoxepin imipramina
20 desipramina 20
0 0 RT [ min] RT [ min] 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Fig. 3 Test Solution TAD 2 Fig. 4 Plasma Calibrator lyophil. R.T. 5.8 Nordoxepin R.T. 6.0 Nordoxepin 150 ug/l R.T. 6.5 Desipramine R.T. 7.0 Desipramine 150 ug/l R.T. 7.6 Imipramine R.T. 7.8 Imipramine 150 ug/l R.T. 10.0 Internal Standard R.T. 10.0 Internal Standard
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